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The effect of dietary TAG structure and fatty acid acyl TAG position on palmitic and linoleic acid metabolism was investigated in four middle-aged male subjects. The study design consisted of feeding diets containing 61 g/d of native lard (NL) or randomized lard (RL) for 28 d. Subjects then received an oral dose of either 1,3-tetradeuteriopalmitoyl-2-dideuteriolinoleoyl-rac-glycerol or a mixture of 1,3-dideuteriolinoleoyl-2-tetradeuteriopalmitoyl-rac-glycerol and 1,3-hexadeuteriopalmitoyl-2-tetradeuteriolinoleoyl-rac-glycerol. Methyl esters of plasma lipids isolated from blood samples drawn over a 2-d period were analyzed by GC-MS. Results showed that absorption of the 2H-fatty acids (2H-FA) was not influenced by TAG position. The 2H-FA at the 2-acyl TAG position were 85+/-4.6% retained after absorption. Substantial migration of 2H-16:0 (31.2+/-8.6%) from the sn-2 TAG position to the sn-1,3 position and 2H-18:2n-6 (52.8+/-6.4%) from the sn-1,3 position to the sn-2 position of chylomicron TAG occurred after initial absorption and indicates the presence of a previously unrecognized isomerization mechanism. Incorporation and turnover of the 2H-FA in chylomicron TAG, plasma TAG, and plasma cholesterol esters were not influenced by TAG acyl position. Accretion of 2H-16:0 from the sn-2 TAG position in 1-acylphosphatidylcholine was 1.7 times higher than 2H-16:0 from the sn-1,3 TAG positions. Acyl TAG position did not influence 2H-18:2n-6 incorporation in PC. The concentration of 2H-18:2n-6-derived 2H-20:4n-6 in plasma PC from subjects fed the RL diet was 1.5 times higher than for subjects fed the NL diet, and this result suggests that diets containing 16:0 located at the sn-2 TAG position may inhibit 20:4n-6 synthesis. The overall conclusion is that selective rearrangement of chylomicron TAG structures diminishes but does not totally eliminate the metabolic and physiological effects of dietary TAG structure.

Background: Treatment of acute myocardial infarction (AMI) is changing, and differences in medical practice are observed within and between countries on the basis of local practice patterns and available technology. These differing approaches provide an opportunity to evaluate medical practice and outcomes at the population level. The primary aim of this study was to compare medical care in patients hospitalized with AMI in 2 large cities in Sweden and the United States. A secondary aim was to compare medical outcomes., Methods: All resident patients (age range, 30-74 years) hospitalized with AMI in Göteborg, Sweden (1995-1996), and a representative population-based sample of all patients with AMI in Minneapolis/St. Paul, Minn (1995)., Results: Patients with AMI in Göteborg (GB) were older than patients in Minneapolis/St. Paul (MSP), but fewer patients in GB had a prior history of cardiovascular disease. During the AMI admission, coronary angiography, percutaneous coronary angioplasty (PTCA), and coronary artery bypass grafting (CABG) were performed twice as frequently in MSP than in GB. Echocardiogram and exercise testing were more frequently performed in GB. During hospitalization, beta-blockers were more frequently prescribed in GB, whereas calcium channel blockers, long- and short-acting nitrates, intravenous nitroglycerine, digitalis, aspirin, oral anticoagulants, heparin, and lidocaine were significantly more common in MSP. Thrombolysis, acute PTCA, ACE inhibitors, and diuretics were similar. Reinfarction was higher in men in GB (4% vs 1%, P <.009) and women in GB (3% vs 1%, P = not significant). On discharge, beta-blockers and diuretics were prescribed significantly more often in GB, whereas calcium channel blockers, nitrates, and digitalis were prescribed more often in MSP. Aspirin and ACE inhibitors had similar usage rates. Despite these diagnostic and treatment contrasts, there were no differences in mortality rate at 30 days or after 3 years of follow-up after risk-adjusting for patient baseline differences., Conclusion: Comparing patients hospitalized with AMI in MSP and GB, we found marked differences in medical care, with invasive strategies more likely to be used in MSP. This may be the result of historical practice patterns, the healthcare system, and healthcare financing differences. Despite these differences, short- or long-term mortality rates were identical.

Over four-hundred crude extracts from 202 plant species distributed among 131 plant families were evaluated for their bioactivity against brine shrimp (Artemia salina). Activity was determined for both the organic (CH2Cl2:MeOH) and aqueous extracts against A. salina in a 96 well-plate assay. Of the greater than four-hundred extracts tested, 21 organic and 6 aqueous extracts demonstrated potent cytotoxic activity (LC50 = < 100 microg/ml). Three of these organic extracts (Crateva religiosa, Diospyros dichrophylla, and Olax subscorpioidea) were chosen for chemical investigations due to their high activity and a lack of prior investigations. Chemical analysis of these extracts resulted in the isolation of oleanolic acid (1) and 4-epi-hederagenin (2) from C. religiosa, isodiospyrin (3) from D. dichrophylla, and santalbic acid (4) from O. subscorpioidea.

The limitations in design and analysis of currently available dose-response studies of SSRI treatment of major depression have led to the conclusion that dose response is flat. We applied concepts from our companion article to determine if currently available data is consistent with a "potential" and an "expressed" dose response. Using these concepts, we performed a meta-analysis on all identifiable published fixed-dose and dose-escalation studies that reported the effect of different SSRI oral doses on efficacy. "Potential" dose response in fixed-dose studies with categorical response outcomes equaled a significant meta-analyzed slope of 3.1%/100 SSRI mg equivalents (SMEs) (SE=1.2%) or 7.8% across the dose range. Similar analysis in dose-escalation studies that reported categorical response data yielded a non-significant meta-analyzed slope of 3.7%/100 SMEs (SE=2.3%) or 9.3% across the dose range. Analyses of the "expressed" dose response demonstrated in the studies indicated a slope statistically equal to zero. The current analysis suggests a "potential" dose response can be demonstrated for SSRIs in treating major depression. The analysis suggests an "expressed" dose response could exist in best clinical practice. Study designs better tailored to address the relevant clinical question would test these hypotheses more appropriately than previous studies., (Copyright 2003 Wiley-Liss, Inc.)

The purpose of this study was to investigate the effect of dietary CLA on accretion of 9c-18:1, 9c,12c-18:2, 10t,12c-18:2, and 9c,11t-18:2 and conversion of these FA to their desaturated, elongated, and chain-shortened metabolites. The subjects were six healthy adult women who had consumed normal diets supplemented with 6 g/d of sunflower oil or 3.9 g/d of CLA for 63 d. A mixture of 10t,2c-18:2-d4, 9c,11t-18:2-d6, 9c-18:1-d8, and 9c,12c-18:2-d2, as their ethyl esters, was fed to each subject, and nine blood samples were drawn over a 48-h period. The results show that dietary CLA supplementation had no effect on the metabolism of the deuterium-labeled FA. These metabolic results were consistent with the general lack of a CLA diet effect on a variety of physiological responses previously reported for these women. The 2H-CLA isomers were metabolically different. The relative percent differences between the accumulation of 9c,11t-18:2-d6 and 10t,12c-18:2-d4 in plasma lipid classes ranged from 9 to 73%. The largest differences were a fourfold higher incorporation of 10t,12c-18:2-d4 than 9c,11t-18:2-d6 in 1-acyl PC and a two- to threefold higher incorporation of 9c,11t-18:2-d6 than 10t,12c-18:2-d4 in cholesterol esters. Compared to 9c-18:1-d8 and 9c,12c-18:2-d2, the 10t,12c-18:2-d4 and 9c,11t-18:2-d6 isomers were 20-25% less well absorbed. Relative to 9c-18:1, incorporation of the CLA isomers into 2-acyl PC and cholesterol ester was 39-84% lower and incorporation of 10t,12c-18:2 was 50% higher in 1-acyl PC. This pattern of selective incorporation and discrimination is similar to the pattern generally observed for trans and cis 18:1 positional isomers. Elongated and desaturated CLA metabolites were detected. The concentration of 6c,10t,12c-18:3-d4 in plasma TG was equal to 6.8% of the 10t,12c-18:2-d4 present, and TG was the only lipid fraction that contained a CLA metabolite present at concentrations sufficient for reliable quantification. In conclusion, no effect of dietary CLA was observed, absorption of CLA was less than that of 9c-18:1, CLA positional isomers were metabolically different, and conversion of CLA isomers to desaturated and elongated metabolites was low.

The purpose of this study was to determine the effectiveness of biofeedback in the treatment of stages 1 and 2 essential hypertension via meta-analytical methods. A utilization-focused integrative review was limited to adult randomized clinical trials, and study groups were categorized into biofeedback, active control, and inactive control. Both biofeedback and active control treatments resulted in a reduction in systolic blood pressure (SBP) and diastolic blood pressure (DBP). Only biofeedback (with related cognitive therapy and relaxation training) showed a significantly greater reduction in both SBP (6.7 mm Hg) and DBP (4.8 mm Hg) when compared with inactive control treatments. Nurses in practice settings should consider biofeedback therapy for their hypertensive clients., (Copyright 2001 by W.B. Saunders Company.)

Objective: To assess the effect of publication bias on the results and conclusions of systematic reviews and meta-analyses., Design: Analysis of published meta-analyses by trim and fill method., Studies: 48 reviews in Cochrane Database of Systematic Reviews that considered a binary endpoint and contained 10 or more individual studies., Main Outcome Measures: Number of reviews with missing studies and effect on conclusions of meta-analyses., Results: The trim and fill fixed effects analysis method estimated that 26 (54%) of reviews had missing studies and in 10 the number missing was significant. The corresponding figures with a random effects model were 23 (48%) and eight. In four cases, statistical inferences regarding the effect of the intervention were changed after the overall estimate for publication bias was adjusted for., Conclusions: Publication or related biases were common within the sample of meta-analyses assessed. In most cases these biases did not affect the conclusions. Nevertheless, researchers should check routinely whether conclusions of systematic reviews are robust to possible non-random selection mechanisms.

We study recently developed nonparametric methods for estimating the number of missing studies that might exist in a meta-analysis and the effect that these studies might have had on its outcome. These are simple rank-based data augmentation techniques, which formalize the use of funnel plots. We show that they provide effective and relatively powerful tests for evaluating the existence of such publication bias. After adjusting for missing studies, we find that the point estimate of the overall effect size is approximately correct and coverage of the effect size confidence intervals is substantially improved, in many cases recovering the nominal confidence levels entirely. We illustrate the trim and fill method on existing meta-analyses of studies in clinical trials and psychometrics.

This paper deals with the reanalysis of serum lipids from previous studies in which deuterated fatty acids were administered to a single person. Samples were reanalyzed to determine if the deuterated fatty acids were converted to deuterium-labeled conjugated linoleic acid (CLA, 9c,11t-18:2) or other CLA isomers. We found 11-trans-octadecenoate (fed as the triglyceride) was converted (delta9 desaturase) to CLA, at a CLA enrichment of ca. 30%. The 11-cis-octadecenoate isomer was also converted to 9c,11c-18:2, but at <10% the concentration of the 11t-18:1 isomer. No evidence (within our limits of detection) for conversion of 10-cis- or 10-trans-octadecenoate to the 10,12-CLA isomers (delta12 desaturase) was found. No evidence for the conversion of 9-cis,12-cis-octadecadienoate to CLA (via isomerase enzyme) was found. Although these data come from four single human subject studies, data from some 30 similar human studies have convinced us that the existence of a metabolic pathway in one subject may be extrapolated to the normal adult population.

The effect of dietary docosahexaenoic acid (22:6n-3, DHA) on the metabolism of oleic, linoleic, and linolenic acids was investigated in male subjects (n = 6) confined to a metabolic unit and fed diets containing 6.5 or <0.1 g/d of DHA for 90 d. At the end of the diet period, the subjects were fed a mixture of deuterated triglycerides containing 18:1n-9[d6], 18:2n-6[d2], and 18:3n-3[d4]. Blood samples were drawn at 0, 2, 4, 6, 8, 12, 24, 48, and 72 h. Methyl esters of plasma total lipids, triglycerides, phospholipids, and cholesterol esters were analyzed by gas chromatography-mass spectrometry. Chylomicron triglyceride results show that the deuterated fatty acids were equally well absorbed and diet did not influence absorption. Compared to the low-DHA diet (LO-DHA), clearance of the labeled fatty acids from chylomicron triglycerides was modestly higher for subjects fed the high DHA diet (HI-DHA). DHA supplementation significantly reduced the concentrations of most n-6[d2] and n-3[d4] long-chain fatty acid (LCFA) metabolites in plasma lipids. Accumulation of 20:5n-3[d4] and 22:6n-3[d4] was depressed by 76 and 88%, respectively. Accumulations of 20:3n-6[d2] and 20:4n-6[d2] were both decreased by 72%. No effect of diet was observed on acyltransferase selectivity or on uptake and clearance of 18:1n-9[d6], 18:2n-6[d2], and 18:3n-3[d4]. The results indicate that accumulation of n-3 LCFA metabolites synthesized from 18:3n-3 in typical U.S. diets would be reduced from about 120 to 30 mg/d by supplementation with 6.5 g/d of DHA. Accumulation of n-6 LCFA metabolites synthesized from 18:2n-6 in U.S. diets is estimated to be reduced from about 800 to 180 mg/d. This decrease is two to three times the amount of n-6 LCFA in a typical U.S. diet. These results support the hypothesis that health benefits associated with DHA supplementation are the combined result of reduced accretion of n-6 LCFA metabolites and an increase in n-3 LCFA levels in tissue lipids.

1. The degree of cell-to-cell coupling between ventricular myocytes of neonatal rats appeared well preserved when studied in the perforated version of the patch clamp technique or, in double whole-cell conditions, when ATP was present in the patch pipette solution. In contrast, when ATP was omitted, the amplitude of junctional current rapidly declined (rundown). 2. To examine the mechanism(s) of ATP action, an 'internal perfusion technique' was adapted to dual patch clamp conditions, and reintroduction of ATP partially reversed the rundown of junctional channels. 3. Cell-to-cell communication was not preserved by a non-hydrolysable ATP analogue (5'-adenylimidodiphosphate, AMP-PNP), indicating that the effect most probably did not involve direct interaction of ATP with the channel-forming proteins. 4. An ATP analogue supporting protein phosphorylation but not active transport processes (adenosine 5'-O-(3-thiotriphosphate), ATPgammaS) maintained normal intercellular communication, suggesting that the effect was due to kinase activity rather than to altered intracellular Ca2+. 5. A broad spectrum inhibitor of endogenous serine/threonine protein kinases (H7) reversibly reduced the intercellular coupling. A non-specific exogenous protein phosphatase (alkaline phosphatase) mimicked the effects of ATP deprivation. The non-specific inhibition of endogenous protein phosphatases resulted in the preservation of substantial cell-to-cell communication in ATP-free conditions. 6. The activity of gap junctional channels appears to require both the presence of ATP and protein kinase activity to counteract the tonic activity of endogenous phosphatase(s).

The influence of dietary supplementation with 20:4n-6 on uptake and turnover of deuterium-labeled linoleic acid (18:2n-6[d2]) in human plasma lipids and the synthesis of desaturated and elongated n-6 fatty acids from 18:2n-6[d21 were investigated in six adult male subjects. The subjects were fed either a high-arachidonic acid (HIAA) diet containing 1.7 g/d or a low-AA (LOAA) diet containing 0.21 g/d of AA for 50 d. Each subject was then dosed with about 3.5 g of 18:2n-6[d2] as the triglyceride (TG) at 8:00 A.M., 12:00, and 5:00 P.M. The total 18:2n-6[d21] fed to each subject was about 10.4 g and is approximately equal to one-half of the daily intake of 18:2n-6 in a typical U.S. male diet. Nine blood samples were drawn over a 96-h period. Methyl esters of plasma total lipid (TL), TG, phospholipid, and cholesterol ester were analyzed by gas chromatography-mass spectroscopy. Dietary 20:4n-6 supplementation did not affect uptake of 18:2n-6[d2] in plasma lipid classes over the 4-d study period nor the estimated half-life of 24-36 h for 18:2n-6[d2]. The percentages of major deuterium-labeled desaturation and elongation products in plasma TL, as a percentage of total deuterated fatty acids, were 1.35 and 1.34% 18:3n-6[d2]; 0.53 and 0.50% 20:2n-6[d2]; 1.80 and 0.92% 20:3n-6[d2] and 3.13 and 1.51% 20:4n-6[d2] for the LOAA and HIAA diet groups, respectively. Trace amounts (<0.1%) of the 22:4n-6[d2] and 22:5n-6[d2] metabolites were present. Plasma TL concentration data for both 20:3n-6[d2] and 20:4n-6[d2] were 48% lower (P < 0.05) in samples from the HIAA diet group than in samples from the LOAA diet group. For a normal adult male consuming a typical U.S. diet, the estimated accumulation in plasma TL of 20:4n-6 synthesized from 20 g/d (68 mmole) of 18:2n-6 is 677 mg/d (2.13 mmole). Dietary supplementation with 1.5 g/d of 20:4n-6 reduced accumulation of 20:4n-6 synthesized from 20 g/d of 18:2n-6 to about 326 mg/d (1.03 mmole).

Objective: To test the hypothesis that mild hypothermia potentiates the cardioprotection afforded by ischaemic preconditioning so that infarct size limitation can be obtained after coronary artery occlusion (CAO) durations which exceed the cardioprotective range (> 90 min) of either hypothermia or ischaemic preconditioning alone., Methods: Four groups of anaesthetized rats were subjected to different durations of CAO: (i) normothermia (N, 36.5-37.5 degrees C, n = 29), (ii) normothermia + ischaemic preconditioning (N + IP, 15 min CAO followed by 10 min of reperfusion, n = 35), (iii) hypothermia (H, 30-31 degrees C, n = 31) and (iv) hypothermia + ischaemic preconditioning (H + IP, n = 24). Infarct size (IA/AR) was determined after 3 hours of reperfusion using trypan blue to delineate the area at risk (AR) from non-risk region and nitroblue tetrazolium to delineate infarcted area (IA) from viable myocardium., Results: In N the CAO duration versus infarct size relation had a sigmoid shape with virtually no infarction occurring at 15 min CAO and 56 +/- 5% of the area at risk being infarcted at 30 min CAO reaching a plateau of 71 +/- 2% at 60 min CAO. Hypothermia produced a rightward shift of the relation resulting in an approximately 15 min delay in onset of infarction. Ischaemic preconditioning produced a similar reduction in infarct size (23 +/- 4%) at 30 min CAO compared to hypothermia (13 +/- 3%) but also limited infarct size at 45 min to 36 +/- 3% and at 60 min CAO to 50 +/- 3% suggesting a slowing of infarct progression. Neither intervention limited IA/AR produced by 120 min CAO. In H + IP, combined hypothermia and ischaemic preconditioning resulted in synergistic infarct size reduction so that at 45 min and 60 min CAO IA/AR was reduced to 17 +/- 3% and 23 +/- 3%, respectively, and even at 120 min CAO to 58 +/- 5%, which was significantly smaller than during normothermic control conditions (p < 0.05 vs. N)., Conclusion: Mild hypothermia limited IA/AR modestly but markedly enhanced the cardioprotection afforded by ischaemic preconditioning in the in situ rat heart so that irreversible damage produced by even prolonged coronary artery occlusions was limited.

This study investigated the influence of dietary arachidonic acid (20:4n-6) on delta 5 desaturation and incorporation of deuterium-labeled 8cis,11 cis, 14-eicosatrienoic acid (20:3n-6) into human plasma lipids. Adult male subjects (n = 4) were fed diets containing either 1.7 g/d (HI20:4 diet) or 0.21 g/d (LO20:4 diet) of arachidonic acid for 50 d and then dosed with a mixture containing ethyl esters of 20:3n-6[d4] and 18:1n-9[d2]. A series of blood samples was sequentially drawn over a 72-h period, and methyl esters of plasma total lipid, triacylglycerol, phospholipids, and cholesteryl ester were analyzed by gas chromatography-mass spectrometry. Based on the concentration of 20:3n6[d4] in total plasma lipid, the estimated conversion of 20:3n-6[d4] to 20:4n-6[d4] was 17.7 +/- 0.79% (HI20:4 diet) and 2.13 +/- 1.44% (LO20:4 diet). The concentrations of 20:4n-6[d4] in total plasma lipids from subjects fed the HI20:4 and LO20:4 diets were 2.10 +/- 0.6 and 0.29 +/- 0.2 mumole/mL plasma/mmole of 20:3n-6[d4] fed/kg of body weight. These data indicate that conversion of 20:3n-6[d4] to 20:4n-6[d4] was stimulated 7-8-fold by the HI20:4 diet. Phospholipid acyltransferase was 2.5-fold more selective for 20:3n-6[d4] than 18:1n-9[d2], and lecithin:cholesterol acyltransferase was 2-fold more selective for 18:1n-9[ds] than 20:3n-6[d4]. These differences in selectivity were not significantly influenced by diet. Absorption of ethyl 20:3n-6[d4] was about 33% less than ethyl 18:1n-9[d2]. The sum of the n-6 retroconversion products from 20:3n-6[d4] in total plasma lipids was about 2% of the total deuterated fatty acids. Neither absorption nor retroconversion appears to be influenced by diet.

Cultured C6 glioma cells rapidly incorporate and metabolize the essential fatty acids, 18:2(n-6) and 18:3(n-3), to 20- and 22-carbon polyunsaturated fatty acids. Using several deuterated fatty acid substrates we have obtained data that suggest alternate pathways, one possibly involving delta 8-desaturation, may exist in glioma cells for formation of 20:5(n-3) and 22:6(n-3) from 18:3(n-3). With 18:3(n-3)-6,6,7,7-d4 practically no 18:4(n-3)-6,7-d2 or 20:4(n-3)-8,9-d2 was detected whereas 20:3(n-3)-8,8,9,9-d4 accounted for 3.4% and delta 5,11,14,17-20:4-8,8,9,9-d4 for 21.1% of the total deuterated fatty acids recovered in phospholipids after a 16 h incubation; 20:5(n-3)-8,9-d2, 22:5(n-3)-10,11-d2, and 22:6(n-3)-10,11-d2 accounted for 42.4%, 13.2%, and 2.8% of deuterated acyl chains, respectively. When added exogneously, 20:3-8,8,9,9,-d4 was extensively converted to delta 5,11,14,17-20:4(n-3)-8,8,9,9-d4 (45%) and 20:5(n-3)-8,9-d2 (24%); a small amount (4%) of 18:3(n-3)-d4 also was detected. Both 20:4(n-3)-8,9-d2 and 18:4(n-3)-12,13,15,16-d4 were also converted to 20:5(n-3) and 22:6(n-3) with 8 and 0% of the respective original deuterated substrate remaining after 16 h. A possible pathway for 18:3(n-3) metabolism in glioma cells is described whereby an initial chain elongation step is followed by successive delta 5 and delta 8 desaturation reactions resulting in 20:5(n-3) formation and accounting for the ordered removal of deuterium atoms. Alternatively, extremely effective retroconversion may occur to chain shorten 20:3(n-3)-d4 to 18:3(n-3)-d4 followed by rapid conversion through the classical desaturation and chain elongation sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

An analytical method that was developed to analyze deuterium-labeled fatty acids in human blood has been extended to identify labeled fatty acids from C14 to C24 chain length which are formed by metabolic processes such as desaturation, elongation, or shortening of the labeled fatty acids fed. A new computer and a hardware adder have been utilized to assure reliable data acquisition. Relative standard deviations for the analysis of labeled fatty acids were measured at 0.02, 0.03, and 0.04 at the 5%, 1%, and 0.2% levels of the labeled fatty acid methyl esters, respectively. The method makes extensive use of standards and computer processing for accuracy and high productivity. Data from a chylomicron triacylglycerol fraction are included to demonstrate the sensitivity of detection of metabolites formed by desaturation and elongation.

An unusual Ph 1 translocation in a woman, 65 years old, with chronic myelocytic leukemia is reported. Cytogenetic analysis performed on 150 mitoses examined with Q-, R-, and C-banding revealed evidence for an unusual translocation involving the insertion of a segment of chromosome #9 into chromosome #22, thus masking the presence of a Ph 1. The anomaly can best be described as 46, XX, ins(22;9) (q11;q22 to 34).

Fortified milks were saponified overnight at room temperature with 1% ethanolic pyrogallol and KOH. The digest was extracted with hexane after adding water and ethanol, and the extract was washed consecutively with 5% KOH, water, and 55% aqueous ethanol to remove polar lipids. After evaporation, the residue was first chromatographed on a column of 5 micrometer silica. A fraction containing vitamin D was collected, evaporated, and rechromatographed on a reverse phase column for the separation and quantitation of vitamins D2 and D3. Recovery was 96-99% and the coefficient of variation was 3% (8 replicates). Infant formula was diluted and then saponified and extracted as in the analysis of milk. Margarine was saponified by shaking overnight with 1% ethanolic pyrogallol and 80% KOH. Water and ethanol were added to the digest before extraction. Extracts from formula and margarine were chromatographed as milk except, before HPLC, the extract was dissolved in isopropanol-hexane (1 + 99) and passed through 5 cm alumina in a Pasteur pipet, and the concentration of isopropanol in the first high performance liquid chromatographic (HPLC) solvent system was halved to improve the separation of vitamin D from other absorbing lipids. Usually several peaks were obtained during the final HPLC analysis, and the identification of vitamins D2 and D3 was less certain than in the analysis of milk. The coefficients of variation for formula and margarine were 6% (5 replicates) and 9% (6 replicates), respectively.